TW202143998A - Method and medicine for treating huntington's disease - Google Patents

Abstract

Provided are a method and medicine for treating Huntington's disease, comprising administering to a subject a therapeutically effective amount of a component of a plasminogen activation pathway or a related compound thereof. Also provided are a medicine containing the component or the compound for treating Huntington's disease, a pharmaceutical composition, an article of manufacture, and a kit.

Description

A method and drug for the treatment of Huntington's disease

The present invention relates to the treatment of Huntington's disease, comprising administering to a subject an effective amount of a component of the plasminogen activation pathway or a related compound thereof, such as plasminogen, to treat Huntington's disease.

Huntington's disease (HD), also known as major chorea or Huntington's chorea, is an autosomal dominant neurodegenerative disease. The causative gene is the IT-15 gene (also known as the HTT gene) located on chromosome 4p16.3, which has abnormal expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in Huntington's disease patients. When the number of copies is greater than 40 times, it has full penetrance. Generally, the onset of the disease occurs in middle age, and the main symptoms include progressive dyskinesia, cognitive decline and psychiatric symptoms typically manifested by chorea-like symptoms. At present, the second-generation antipsychotics such as tetrabenazine or olanzapine are often used to control chorea symptoms; antidepressants are often used to improve mental symptoms such as depression. However, these drugs do not delay the progression of the disease. At present, there are also some studies actively exploring the cause of disease treatment through gene therapy, but there are many uncertainties in the method of gene therapy. Therefore, how to find another way to find more effective treatment methods has become the focus of research on this disease.

The present study finds that plasminogen can significantly improve Huntington's disease and its related symptoms, such as improving movement disorders in subjects with Huntington's disease, improving cognitive dysfunction, slowing down weight loss, protecting undamaged neurons, and promoting the development of damaged neurons. Repairs and/or improves neuropsychiatric symptoms, providing an effective treatment for Huntington's disease.

The present invention relates to the following:

1. In one aspect, the application relates to a method for the treatment of Huntington's disease, comprising administering to a subject suffering from Huntington's disease a therapeutically effective amount of one or more compounds selected from the group consisting of: a component of a plasminogen activation pathway, capable of directly Compounds that activate plasminogen or indirectly activate plasminogen by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, are capable of upregulating fibrin Antagonists of lysinogen or plasminogen activator-expressed compounds, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and fibrinolysis inhibitors.

In one aspect, the present application relates to the use of one or more compounds selected from the group consisting of components of the plasminogen activation pathway, capable of directly activating fibrin, in the manufacture of a medicament for the treatment of Huntington's disease. Lysinogen or compounds that activate plasminogen indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, are capable of upregulating plasminogen Or plasminogen activator-expressed compounds, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.

In one aspect, the application relates to a medicament or pharmaceutical composition comprising one or more compounds selected from the group consisting of components of the plasminogen activation pathway, capable of direct Compounds that activate plasminogen or indirectly activate plasminogen by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, are capable of upregulating fibrin Antagonists of lysinogen or plasminogen activator-expressed compounds, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and fibrinolysis inhibitors.

2. The method, purposes, medicine or pharmaceutical composition of item 1, wherein the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasmin, Lys-plasminogen. zymogen, Glu-plasminogen, plasmin, plasminogen and plasmin containing one or more kringle domains and protease domains of plasminogen and plasmin Variants and analogs, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activator, tPA and uPA.

3. The method, use, medicament or pharmaceutical composition of item 1, wherein the antagonist of the fibrinolysis inhibitor is an inhibitor of PAI-1, complement C1 inhibitor, α2 antiplasmin or α2 macroglobulin, such as an antibody .

4. The method, use, medicament or pharmaceutical composition of any one of items 1-3, wherein the compound has one or more effects selected from the group consisting of improving or relieving movement disorders, improving cognition on the Huntington's disease subject Dysfunction, slow down weight loss, promote repair of damaged neurons, improve neuropsychiatric symptoms, relieve anxiety, reduce hippocampal GFAP expression, reduce hippocampal apoptosis, promote cerebellar, hippocampal or striatal neuronal Nissl bodies Quantitative recovery, repair of damage to the hippocampus, striatum or olfactory tubercle, promotion of hippocampal or striatal BDNF expression, and promotion of striatal remyelination.

5. The method, use, medicament or pharmaceutical composition of any one of items 1-4, wherein the compound is used in combination with one or more other drugs or methods of treatment.

6. The method, use, medicament or pharmaceutical composition of item 5, wherein the other medicament or method of treatment is selected from one or more of the following: antidopaminergic drugs, dopamine receptor inhibitors, antipsychotics (such as butylated benzene) and phenothiazines), gamma-aminobutyric acid transferase inhibitors, cell transplantation therapy and gene therapy.

7. The method, use, medicament or pharmaceutical composition of any one of items 1-6, wherein the compound is plasminogen.

8. The method, use, medicament or pharmaceutical composition of any one of items 1-7, wherein the plasminogen is human full-length plasminogen or a conservatively substituted variant thereof.

9. The method, use, medicament or pharmaceutical composition of any one of items 1-7, wherein the plasminogen and Sequence 2 have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity and still possess the lysine-binding or proteolytic activity of plasminogen.

10. The method, use, medicament or pharmaceutical composition of any one of items 1-7, said plasminogen comprising at least 80%, 90%, 95%, 96%, 97%, 98%, A protein with an amino acid sequence of 99% amino acid sequence identity and still having the proteolytic activity of plasminogen.

11. The method, use, medicament or pharmaceutical composition of any one of items 1 to 7, wherein the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microfibrils Lysinogen, delta-plasminogen or variants thereof that retain the proteolytic activity of plasminogen.

12. The method, use, medicament or pharmaceutical composition of any one of items 1 to 7, wherein the plasminogen comprises the amino acid sequence shown in sequence 2, 6, 8, 10, 12 or comprises sequence 2, 6, 8 Conservative substitution variants of amino acid sequences shown in , 10 and 12.

13. The method, use, medicament or pharmaceutical composition of any one of items 1-12, wherein the compound is by nasal inhalation, aerosol inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, Subcutaneous, intracranial, intrathecal, intraarterial (eg via the carotid artery) or intramuscular administration.

In any of the above embodiments of the invention, the plasminogen may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97% with sequence 2, 6, 8, 10 or 12 %, 98% or 99% sequence identity and still have plasminogen activity, such as lysine binding activity or proteolytic activity. In some embodiments, the plasminogen is based on sequence 2, 6, 8, 10 or 12 with additions, deletions and/or substitutions of 1-100, 1-90, 1-80, 1-70 , 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1 -3, 1-2, 1 amino acid, and still have plasminogen activity, such as lysine binding activity or proteolytic activity.

In some embodiments, the plasminogen is a protein comprising a fragment of plasminogen activity and still having plasminogen activity, such as lysine binding activity or proteolytic activity. In some embodiments, the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, microplasminogen, microplasminogen, delta-plasminogen, or retention thereof Plasminogen activity, eg, a variant of proteolytic activity. In some embodiments, the plasminogen is natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity, eg, lysine binding activity or proteolytic activity. In some embodiments, the plasminogen is a human plasminogen ortholog from a primate or rodent or it still retains plasminogen activity, such as lysine binding activity or protein Hydrolytically active variants or fragments. In some embodiments, the amino acid of the plasminogen is shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is human native plasminogen.

In some embodiments, the subject is a human. In some embodiments, the subject is deficient or deficient in plasminogen. In some embodiments, the deficiency or deletion is congenital, secondary and/or local.

In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and plasminogen for use in the aforementioned methods. In some embodiments, the kit may be a prophylactic or therapeutic kit comprising: (i) plasminogen for use in the aforementioned methods and (ii) for delivering the plasminogen to the the subjects' means. In some embodiments, the member is a syringe or vial. In some embodiments, the kit further comprises a label or instructions for administering the plasminogen to the subject to perform any of the foregoing methods.

In some embodiments, the article of manufacture comprises: a container comprising a label; and comprising (i) plasminogen or a pharmaceutical composition comprising plasminogen for use in the aforementioned methods, wherein the label indicates that the plasminogen is to be The lysinogen or composition is administered to the subject to perform any of the foregoing methods.

In some embodiments, the kit or article of manufacture further comprises one or more additional components or containers that contain other medicaments.

In some embodiments of the foregoing methods, the plasminogen is treated by systemic or topical administration, preferably by intravenous, intramuscular, subcutaneous administration of plasminogen. In some embodiments of the foregoing methods, the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer. In some embodiments of the foregoing methods, the plasminogen is administered at 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100mg/kg, 10-100mg/kg (per kg body weight) or 0.0001-2000mg/cm2, 0.001-800 mg/cm2, 0.01-600 mg/cm2, 0.1-400 mg/cm2, 1-200 mg /cm2, 1-100 mg/cm2, 10-100 mg/cm2 (calculated on a per square centimeter body surface area), preferably repeated at least once, preferably at least daily.

The present invention explicitly covers all combinations of technical features belonging to the embodiments of the present invention, and the technical solutions after these combinations have been explicitly disclosed in this application, just as the above-mentioned technical solutions have been separately and explicitly disclosed. In addition, the present invention also explicitly covers the combination of each embodiment and its elements, and the technical solution after the combination is explicitly disclosed herein.

Huntington's disease (HD) is a single-gene autosomal dominant neurodegenerative disease whose pathogenesis is mainly mHtt protein toxicity. The main symptoms include progressive aggravating movement disorders, cognitive Functional decline and psychiatric symptoms. When the gene mutation causes an abnormal increase in the number of CAG copies, the amino-terminal (N-terminal) polyglutamine chain (polyQ) of the mutant Htt (mHtt) protein can be extended to form an abnormal conformation including a lamellar structure, resulting in mHtt The protein loses normal function and acquires toxic effects.

The "movement disorders" of Huntington's disease mainly include two aspects, involuntary movements (most commonly chorea) and voluntary movement disorders (including motor incoordination and slow movement). Progressive dyskinesias manifest as sudden, rapid throbbing or twitching of the limbs, face, and trunk that are not known in advance, uncontrollable, or uncontrollable slow movements. On examination, chorea-like involuntary movements and hypotonia can be found. Dance-like involuntary movements are the most prominent features of the disease, most of which begin as brief uncontrollable grimacing, nodding, and finger flexion and extension movements, similar to painless convulsions, but slower and non-stereotyped. As the disease progresses, involuntary movements progressively worsen, typical eyebrow raising and head flexion occur, the head turns when staring at objects, the patient walks unsteadily, the gait is leaping, and the hand posture is constantly changing, and the whole body moves like dance. In the later stages of the disease the patient cannot stand and walk due to involuntary movements of the whole body. When the disease progresses, the impairment of voluntary movement becomes more and more obvious, the movement is clumsy, slow, rigid, unable to maintain complex voluntary movements, and dysphagia, speech hesitation and dysarthria appear. Abnormal eye movements. In the advanced stages of the disease, a stuporous state of immobility of the limbs may occur.

Chorea dyskinesia is typical of adult-onset Huntington's disease. Juvenile-type patients with onset before the age of 20 have immobile myotonia as the main movement disorder, manifesting as myotonia, myoclonus, and in the late stage, opistonia. In addition, unlike adults, approximately 50% of patients with juvenile Huntington's disease have generalized seizures.

The "cognitive dysfunction" of Huntington's disease can appear years before motor symptoms and progress more slowly. Progressive dementia​ is another characteristic of people with Huntington's disease. Dementia has the characteristics of subcortical dementia in the early stage, and then manifests as mixed cortical and subcortical dementia in the later stage.

Cognitive impairment can appear in the early stages of Huntington's disease. It begins with a decline in memory and computing abilities in daily life and at work, with only mild impairments in remembering new information, but significant deficits in recall. Changes in speech, including poor oral fluency, mild difficulty finding words, and dysarthria. Impairment of oral fluency is one of the earliest cognitive impairments in Huntington's disease. In the middle and late stages of the disease, patients were unable to complete naming tests that required recall of infrequent words. Choreoid dyskinesia often affects the tongue and lips, disrupts the rhythm and agility of pronunciation, and hinders the volume, speed, rhythm, and length of phrases of speech. Articulation and rhythm disturbances are therefore prominent features of patients with this disorder.

The "neuropsychiatric symptoms" of Huntington's disease can appear very early, even as first symptoms, such as depression, irritability, and apathy, and more severe symptoms include delusional or schizophrenia-like symptoms.

Fibrinolytic system, also known as fibrinolytic system, is a system composed of a series of chemical substances involved in fibrinolysis (fibrinolysis) process, mainly including plasminogen (plasminogen), plasmin , plasminogen activator, fibrinolysis inhibitor. Plasminogen activators include tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). t-PA is a serine protease that is synthesized by vascular endothelial cells. t-PA activates plasminogen, which is mainly carried out on fibrin; urokinase-type plasminogen activator (u-PA) is produced by renal tubular epithelial cells and vascular endothelial cells and can directly activate plasminogen without the need for fibrin as a cofactor. Plasminogen (PLG) is synthesized by the liver. When blood coagulates, a large amount of PLG is adsorbed on the fibrin network, and under the action of t-PA or u-PA, it is activated as plasmin, which promotes fibrinolysis. Plasmin (PL) is a serine protease whose functions are as follows: degrade fibrin and fibrinogen; hydrolyze various coagulation factors V, VIII, X, VII, XI, II, etc.; convert plasminogen into plasmin; Hydrolyzed complement, etc. Fibrinolytic inhibitors: including plasminogen activator inhibitor (PAI) and α2 antiplasmin (α2-AP). PAI mainly has two forms, PAI-1 and PAI-2, which can specifically bind to t-PA in a ratio of 1:1, thereby inactivating it and activating PLG at the same time. α2-AP is synthesized by the liver and combines with PL in a ratio of 1:1 to form a complex, which inhibits the activity of PL; FⅩⅢ makes α2-AP covalently bound to fibrin, reducing the sensitivity of fibrin to PL. Substances that inhibit the activity of the fibrinolytic system in vivo: PAI-1, complement C1 inhibitor; α2 antiplasmin; α2 macroglobulin.

The term "component of the plasminogen activation pathway" of the present invention encompasses:

1. plasminogen, Lys-plasminogen, Glu-plasminogen, micro-plasminogen, delta-plasminogen; variants or analogs thereof;

2. plasmin and their variants or analogs; and

3. Plasminogen activators, such as tPA and uPA, and tPA or uPA variants and analogs comprising one or more domains of tPA or uPA, such as one or more kringle domains and proteolytic domains .

"Variants" of the above-mentioned plasminogen, plasmin, tPA and uPA include all naturally occurring human genetic variants as well as other mammalian forms of these proteins, as well as by additions, deletions and/or substitutions such as 1- 100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acid protein that still has plasminogen, plasmin, tPA or uPA activity. For example, "variants" of plasminogen, plasmin, tPA, and uPA include, for example, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1- 45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 conservative Mutant variants of these proteins obtained by amino acid substitutions.

A "plasminogen variant" of the invention encompasses at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with sequence 2, 6, 8, 10 or 12 % sequence identity and still possess plasminogen activity, such as lysine binding activity or proteolytic activity. For example, a "plasminogen variant" of the present invention may be based on sequence 2, 6, 8, 10 or 12 with additions, deletions and/or substitutions of 1-100, 1-90, 1-80, 1- 70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acid and still have plasminogen activity, such as lysine binding activity or proteolytic activity. Specifically, the plasminogen variants of the present invention include all naturally occurring human genetic variants as well as other mammalian forms of these proteins, as well as by conservative amino acid substitutions such as 1-100, 1-90, 1-80, 1- 70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acid obtained mutant variants of these proteins.

The plasminogen of the invention may be a human plasminogen ortholog from a primate or rodent or a variant thereof that still retains plasminogen activity, such as lysine binding activity or proteolytic activity such as plasminogen shown in sequence 2, 6, 8, 10 or 12, such as human native plasminogen shown in sequence 2.

The aforementioned "analogs" of plasminogen, plasmin, tPA, and uPA include compounds that provide substantially similar effects to plasminogen, plasmin, tPA, or uPA, respectively.

The above-mentioned "variants" and "analogs" of plasminogen, plasmin, tPA and uPA encompass fibers comprising one or more domains (eg, one or more kringle domains and proteolytic domains) "Variants" and "analogs" of lysinogen, plasmin, tPA and uPA. For example, "variants" and "analogs" of plasminogen encompass plasminogen comprising one or more plasminogen domains (eg, one or more kringle domains and proteolytic domains) Variants and analogs such as mini-plasminogen. "Variants" and "analogs" of plasmin encompass "variants" of plasmin comprising one or more plasmin domains (eg, one or more kringle domains and proteolytic domains) and "analogs" such as mini-plasmin and delta-plasmin.

Whether a "variant" or "analog" of the above-mentioned plasminogen, plasmin, tPA or uPA has the activity of plasminogen, plasmin, tPA or uPA, respectively, or does it provide Substantially similar effects of plasminogen, plasmin, tPA or uPA can be detected by methods known in the art, for example, by methods based on enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS ( Fluorescence-activated cell sorting method) is measured by the level of activated plasmin activity, which can be measured, for example, with reference to a method selected from the following documents: Ny, A., Leonardsson, G., Hagglund, AC, Hagglof, P ., Ploplis, VA, Carmeliet, P. and Ny, T. (1999). Ovulation inplasminogen-deficient mice. Endocrinology 140, 5030-5035; Silverstein RL, Leung LL, Harpel PC, Nachman RL (November 1984). "Complex Formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator". J. Clin. Invest. 74 (5): 1625–33; Gravanis I, Tsirka SE (February 2008). "Tissue-type plasminogen activator as a therapeutic target in stroke". Expert Opinion on Therapeutic Targets. 12 (2): 159–70; Geiger M, Huber K, Wojta J, Stingl L, Espana F, Griffin JH, Binder BR (Aug 1989). "Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo". Blood. 74 (2): 722–8.

In some embodiments of the invention, the "component of the plasminogen activation pathway" of the invention is plasminogen. In some embodiments, the plasminogen is human full-length plasminogen or a conservative substitution variant thereof that retains plasminogen activity (eg, its lysine binding activity and proteolytic activity). In some embodiments, the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, microplasminogen, microplasminogen, delta-plasminogen, or retention thereof Variants of plasminogen activity such as its lysine-binding or proteolytic activity. In some embodiments, the plasminogen is natural or synthetic human plasminogen, or a conservative substitution variant thereof that still retains plasminogen activity (eg, its lysine-binding activity or proteolytic activity) or fragments thereof. In some embodiments, the plasminogen is a human plasminogen ortholog from a primate or rodent or a conservatively substituted variant or fragment thereof that still retains plasminogen activity. In some embodiments, the plasminogen comprises the amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 10, or 12. In some embodiments, the plasminogen comprises a conservative substitution sequence of the amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 10, or 12. In some embodiments, the amino acid of the plasminogen is shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is a conservative substitution variant of plasminogen set forth in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is human native plasminogen or a conservative mutant thereof. In some embodiments, the plasminogen is human native plasminogen as shown in SEQ ID NO: 2 or a conservatively substituted variant thereof.

"Compounds capable of directly activating plasminogen or indirectly activating plasminogen by activating upstream components of the plasminogen activation pathway" refers to activating plasminogen either directly or by activating plasminogen Any compound that activates upstream components of the pathway and indirectly activates plasminogen, such as tPA, uPA, streptokinase, saruplase, alteplase, reteplase, tenecteplase, anistreplase, Monteplase, lanoteplase, paamiplase, staphylokinase.

The "antagonist of a fibrinolysis inhibitor" of the present invention is a compound that antagonizes, weakens, blocks, or prevents the action of a fibrinolysis inhibitor. Such fibrinolytic inhibitors are eg PAI-1, complement C1 inhibitor, alpha2 antiplasmin and alpha2 macroglobulin. Such antagonists such as PAI-1, complement C1 inhibitor, α2 antiplasmin or α2 macroglobulin antibodies, or block or downregulate such as PAI-1, complement C1 inhibitor, α2 antiplasmin or α2 macroglobulin Antisense RNA or small RNA expressed by globulin, or occupying the binding site of PAI-1, complement C1 inhibitor, α2 antiplasmin, or α2 macroglobulin but without PAI-1, complement C1 inhibitor, α2 antifibrinolytic A compound that functions as a lysin or alpha2 macroglobulin", or a compound that blocks the binding and/or active domains of PAI-1, complement C1 inhibitor, alpha2 antiplasmin, or alpha2 macroglobulin.

Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease capable of hydrolyzing several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans. In addition, plasmin can activate some metalloproteinase precursors (pro-MMPs) to form active metalloproteinases (MMPs). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis. Plasmin is formed by proteolysis of plasminogen by two physiological PAs: tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). Due to the relatively high levels of plasminogen in plasma and other body fluids, it has traditionally been thought that the regulation of the PA system is mainly achieved through the synthesis and activity levels of PAs. The synthesis of PA system components is tightly regulated by different factors, such as hormones, growth factors and cytokines. In addition, there are specific physiological inhibitors of plasmin and PAs. The main inhibitor of plasmin is α2-antiplasmin (α2-antiplasmin). The activity of PAs was inhibited by both uPA and tPA plasminogen activator inhibitor-1 (PAI-1) and lysinogen activator inhibitor-2 (PAI-2), which mainly inhibited uPA. Certain cell surfaces have uPA-specific cell surface receptors (uPARs) with direct hydrolytic activity.

Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa. Plasminogen is mainly synthesized in the liver and is abundantly present in the extracellular fluid. Plasminogen content in plasma is approximately 2 μM. Plasminogen is thus a huge potential source of proteolytic activity in tissues and body fluids. Plasminogen exists in two molecular forms: glutamate-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen). The naturally secreted and uncleaved form of plasminogen has an amino-terminal (N-terminal) glutamate and is therefore called glutamate-plasminogen. However, in the presence of plasmin, glutamate-plasminogen is hydrolyzed at Lys76-Lys77 to lysine-plasminogen. Compared with glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PAs at a higher rate. The Arg560-Val561 peptide bond of these two forms of plasminogen can be cleaved by either uPA or tPA, resulting in the formation of the disulfide-linked two-chain protease plasmin. The amino-terminal part of plasminogen contains five homologous kringles, so-called kringles, and the carboxy-terminal part contains the protease domain. Some kringles contain lysine-binding sites that mediate the specific interaction of plasminogen with fibrin and its inhibitor α2-AP. Recently, a 38 kDa fragment of plasminogen, including kringles1-4, was found to be a potent inhibitor of angiogenesis. This fragment, named angiostatin, is produced by the hydrolysis of plasminogen by several proteases.

The main substrate of plasmin is fibrin, and the dissolution of fibrin is the key to preventing pathological thrombosis. Plasmin also has substrate specificity for several components of the ECM, including laminin, fibronectin, proteoglycans, and gelatin, suggesting that plasmin also plays an important role in ECM remodeling. Indirectly, plasmin can also degrade other components of the ECM, including MMP-1, MMP-2, MMP-3 and MMP-9, by converting certain protease precursors into active proteases. Therefore, it has been proposed that plasmin may be an important upstream regulator of extracellular proteolysis. In addition, plasmin has the ability to activate certain latent forms of growth factors. In vitro, plasmin also hydrolyzes components of the complement system and releases chemotactic complement fragments.

"Plasmin" is a very important enzyme present in the blood that hydrolyzes fibrin clots into fibrin degradation products and D-dimers.

"Plasminogen" is the zymogen form of plasmin. According to the sequence in swiss prot, it is composed of 810 amino acids according to the natural human plasminogen amino acid sequence (sequence 4) containing the signal peptide, and the molecular weight is about 90kD, a glycoprotein mainly synthesized in the liver and able to circulate in the blood, the cDNA sequence encoding the amino acid sequence is shown in sequence 3. Full-length plasminogen contains seven domains: a C-terminal serine protease domain, an N-terminal Pan Apple (PAp) domain, and five Kringle domains (Kringle 1-5). Referring to the sequence in swiss prot, its signal peptide includes residues Met1-Gly19, PAp includes residues Glu20-Val98, Kringle1 includes residues Cys103-Cys181, Kringle2 includes residues Glu184-Cys262, Kringle3 includes residues Cys275-Cys352, Kringle4 Residues Cys377-Cys454 are included and Kringle5 includes residues Cys481-Cys560. According to NCBI data, the serine protease domain includes residues Val581-Arg804.

Glu-plasminogen is a natural full-length human plasminogen, consisting of 791 amino acids (without a signal peptide of 19 amino acids). The cDNA sequence encoding this sequence is shown in sequence 1, and its amino acid sequence is shown in sequence 2 shown. In vivo, there is also a Lys-plasminogen formed by hydrolysis of amino acids 76-77 of Glu-plasminogen, as shown in sequence 6, and the cDNA sequence encoding this amino acid sequence is shown in sequence 5 Show. Delta-plasminogen (δ-plasminogen) is a fragment of full-length plasminogen that lacks the Kringle2-Kringle5 structure, and only contains Kringle1 and a serine protease domain (also known as a protease domain (PD)), which has been reported in the literature The amino acid sequence of delta-plasminogen (SEQ ID NO: 8) is shown, and the cDNA sequence encoding the amino acid sequence is shown in sequence 7. Mini-plasminogen is composed of Kringle5 and serine protease domains, and it has been reported that it includes residues Val443-Asn791 (with the Glu residue of the Glu-plasminogen sequence without the signal peptide as the starting amino acid) ), its amino acid sequence is shown in sequence 10, and the cDNA sequence encoding the amino acid sequence is shown in sequence 9. Micro-plasminogen (Micro-plasminogen) contains only a serine protease domain, and it has been reported that its amino acid sequence includes residues Ala543-Asn791 (starting from the Glu residues of the Glu-plasminogen sequence without the signal peptide). starting amino acid), there are also patent documents CN102154253A reported that its sequence includes residues Lys531-Asn791 (with the Glu residue of the Glu-plasminogen sequence that does not contain the signal peptide as the starting amino acid), in this patent application, microfibrinolysis The zymogen sequence is referred to the patent document CN102154253A, its amino acid sequence is shown in sequence 12, and the cDNA sequence encoding the amino acid sequence is shown in sequence 11.

The structure of full-length plasminogen is also described in the article by Aisina et al. (Aisina RB, Mukhametova LI. Structure and function of plasminogen/plasmin system[J]. Russian Journal of Bioorganic Chemistry, 2014, 40(6):590-605) middle. In this article, Aisina et al. describe that plasminogen includes Kringle1, 2, 3, 4, 5 domains and a serine protease domain (also known as a protease domain (PD)), in which Kringles is responsible for plasmin The protease binds to both low and high molecular weight ligands (i.e., lysine-binding activity), causing plasminogen to transform into a more open conformation that is more easily activated; the protease domain (PD) is residue Val562 -Asn791, tPA and UPA specifically cleaves the Arg561-Val562 activating bond of plasminogen, thereby allowing plasminogen to form plasmin, therefore, the protease domain (PD) is responsible for conferring plasminogen proteolytic activity area. In the present invention, "plasmin" can be used interchangeably with "plasmin" and "plasminase", and have the same meaning; "plasminogen" is used with "plasminogen" and "plasminogen" " are used interchangeably with the same meaning.

In the present application, the meaning or activity of "deficiency" of plasminogen means that the content of plasminogen in a subject is lower than that of a normal person, and is sufficiently low to affect the normal physiological function of the subject; the The meaning or activity of plasminogen "deletion" is that the content of plasminogen in the subject is significantly lower than that of normal people, or even the activity or expression is extremely low, and normal physiological functions can only be maintained by external supply.

Those with ordinary knowledge in the art can understand that all technical solutions for plasminogen of the present invention are applicable to plasmin, therefore, the technical solutions described in the present invention cover plasminogen and plasmin. During circulation, plasminogen adopts a closed, inactive conformation, but when bound to a thrombus or cell surface, it is converted to an open state mediated by plasminogen activator (PA) Conformation of active plasmin. Active plasmin can further hydrolyze the fibrin clot into fibrin degradation products and D-dimer, thereby dissolving the thrombus. The PAp domain of plasminogen contains an important determinant for maintaining plasminogen in an inactive closed conformation, while the KR domain can bind to lysine residues present on receptors and substrates. A variety of enzymes are known to act as plasminogen activators, including: tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Harger et al. Mann factor) etc.

A "plasminogen-active fragment" refers to the activity of binding to lysine in the target sequence of the substrate (lysine-binding activity), or the activity of exerting a proteolytic function (proteolytic activity), or both proteolytic activity and lysine binding activity. Fragments with amino acid binding activity. The technical solution related to plasminogen of the present invention covers the technical solution of replacing plasminogen with an active fragment of plasminogen. In some embodiments, the plasminogen active fragments described herein comprise or consist of the serine protease domain of plasminogen. In some embodiments, the plasminogen active fragments described herein comprise sequence 14, or comprise at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity to sequence 14 The amino acid sequence of , or consists of sequence 14, or consists of an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identical to sequence 14. In some embodiments, the plasminogen active fragments of the present invention comprise a region selected from one or more of Kringle 1, Kringle 2, Kringle 3, Kringle 4, Kringle 5 or conservative substitution variants thereof, or consist of It consists of regions selected from one or more of Kringle 1, Kringle 2, Kringle 3, Kringle 4, Kringle 5 or conservative substitution variants thereof. In some embodiments, the plasminogen of the present invention includes a protein comprising the active fragment of plasminogen described above.

At present, the methods for measuring plasminogen and its activity in blood include: detection of tissue plasminogen activator activity (t-PAA), detection of plasma tissue plasminogen activator antigen (t-PAAg), Detection of plasma tissue plasminogen activity (plgA), detection of plasma tissue plasminogen antigen (plgAg), detection of plasma tissue plasminogen activator inhibitor activity, plasma tissue plasminogen activator inhibitor Antigen detection and plasma plasmin-antiplasmin complex detection (PAP). The most commonly used detection method is the chromogenic substrate method: adding streptokinase (SK) and a chromogenic substrate to the test plasma, the PLG in the test plasma is converted into PLM under the action of SK, and the latter acts on the hair Chromosomal substrates, which are subsequently measured spectrophotometrically, increase in absorbance proportional to plasminogen activity. In addition, immunochemical methods, gel electrophoresis, immunoturbidimetry, radioimmunoassay, etc. can also be used to measure the plasminogen activity in blood.

"Orthologs or orthologs" refer to homologs between different species, including both protein homologs and DNA homologs, also known as orthologs and vertical homologs. It specifically refers to proteins or genes that have evolved from the same ancestral gene in different species. The plasminogen of the present invention includes human natural plasminogen, and also includes plasminogen orthologs or orthologs derived from different species and having plasminogen activity.

A "conservative substitution variant" refers to one in which a given amino acid residue changes but does not alter the overall conformation and function of the protein or enzyme, this includes, but is not limited to, substitution with similar properties (eg, acidic, basic, hydrophobic, etc.) replace amino acids in the amino acid sequence of the parent protein. Amino acids with similar properties are well known. For example, arginine, histidine and lysine are hydrophilic basic amino acids and are interchangeable. Likewise, isoleucine is a hydrophobic amino acid and can be replaced by leucine, methionine or valine. Therefore, the similarity of two proteins or amino acid sequences of similar functions may differ. For example, 70% to 99% similarity (identity) based on the MEGALIGN algorithm. "Conservative substitution variants" also include polypeptides or enzymes determined to have more than 60% amino acid identity by BLAST or FASTA algorithm, if it can reach more than 75%, it is better, preferably more than 85%, or even more than 90%. is optimal and has the same or substantially similar properties or functions as the native or parent protein or enzyme.

"Isolated" plasminogen refers to a plasminogen protein that has been isolated and/or recovered from its natural environment. In some embodiments, the plasminogen is purified (1) to greater than 90%, greater than 95%, or greater than 98% purity (by weight), as determined by Lowry's method, eg, greater than 99% (by weight), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a spinning cup sequencer, or (3) to homogeneity as determined by the use of Coomassie Determined by Sodium Dodecyl Sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions by thriving blue or silver staining. Isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering techniques and isolated by at least one purification step.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term includes fusion proteins including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences (with or without N-terminal methionine residues); and the like.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as, after gaps have been introduced as necessary to achieve maximum percent sequence identity, and no conservative substitutions are considered part of the sequence identity, the Percentage of amino acid residues that are identical to amino acid residues in a reference polypeptide sequence. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for the purposes of the present invention, percent amino acid sequence identity values were generated using the sequence comparison computer program ALIGN-2.

Where ALIGN-2 is used to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or can be expressed as having or comprising relative to, with, or against a given amino acid sequence A given amino acid sequence A) of a certain % amino acid sequence identity of B is calculated as follows:

Fraction X/Y times 100

where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's A and B alignments, and where Y is the total number of amino acid residues in B. It should be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A with respect to B will not equal the % amino acid sequence identity of B with respect to A. Unless expressly stated otherwise, all % amino acid sequence identity values used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

As used herein, the term "treating" refers to obtaining a desired pharmacological and/or physiological effect. The effect may be complete or partial prevention of the onset, onset of the disease or its symptoms, partial or complete alleviation of the disease and/or its symptoms, and/or partial or complete cure of the disease and/or its symptoms, including: (a) prevention of the disease Occurs or attacks in a subject who may have a predisposition to the disease, but has not been diagnosed with the disease; (b) inhibits the disease, ie, blocks its development; and (c) alleviates the disease and/or its symptoms , that is, causing the disease and/or its symptoms to subside or disappear.

The terms "individual", "subject" and "patient" are used interchangeably herein to refer to mammals including, but not limited to, murine (rat, mouse), non-human primate, human, canine, feline, Hoofed animals (eg horses, cattle, sheep, pigs, goats) etc.

A "therapeutically effective amount" or "effective amount" refers to a component of the plasminogen activation pathway or a component thereof sufficient to effect said prevention and/or treatment of a disease when administered to a mammal or other subject to treat the disease Amounts of related compounds (eg, plasminogen). A "therapeutically effective amount" will vary depending on the component of the plasminogen activation pathway or its related compounds (eg, plasminogen) used, the severity of the disease and/or its symptoms, and the age of the subject to be treated , weight, etc.

Preparation of plasminogen of the present invention

Plasminogen can be isolated from nature and purified for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When the polypeptide is synthesized chemically, the synthesis can be carried out via liquid phase or solid phase. Solid-phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence, is a suitable method for chemical synthesis of plasminogen. Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen. Techniques for solid-phase synthesis are described in Barany and Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Volume 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J . Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12:723-8. Briefly, small insoluble porous beads are treated with functional units on which peptide chains are built. After repeated cycles of coupling/deprotection, the attached solid-phase free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected to reveal new N-terminal amines that can be attached to other amino acids. The peptide remains immobilized on the solid phase, after which it is cleaved off.

Plasminogen of the present invention can be produced using standard recombinant methods. For example, a nucleic acid encoding plasminogen is inserted into an expression vector operably linked to regulatory sequences in the expression vector. Expression control sequences include, but are not limited to, promoters (eg, naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Expression control may be a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells (eg, COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of plasminogen.

Suitable expression vectors typically replicate in the host organism as episomes or as an integral part of the host chromosomal DNA. Typically, the expression vector contains a selectable marker (eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate transformation of the exogenous with the desired DNA sequence of those cells were detected.

Escherichia coli is an example of a prokaryotic host cell that can be used to replicate a subject antibody-encoding polynucleotide. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis and other enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas (Pseudomonas) species. In these prokaryotic hosts, expression vectors can also be generated, which will typically contain expression control sequences (eg, origins of replication) that are compatible with the host cell. In addition, there are many well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from bacteriophage lambda. A promoter will typically control expression, optionally in the case of operator sequences, and have ribosome binding site sequences, etc., to initiate and complete transcription and translation.

Other microorganisms, such as yeast, can also be used for expression. Yeast (eg, S. cerevisiae) and Pichia are examples of suitable yeast host cells, with suitable vectors having expression control sequences (eg, promoters), origins of replication, termination sequences, etc., as desired. Typical promoters contain 3-phosphoglycerate kinase and other saccharolytic enzymes. Inducible yeast are initiated from promoters that include, inter alia, alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

In addition to microorganisms, mammalian cells (eg, mammalian cells grown in in vitro cell culture) can also be used to express and produce the anti-Tau antibodies of the invention (eg, polynucleotides encoding the subject anti-Tau antibodies). See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for use in these cells may contain expression control sequences, such as origins of replication, promoters and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), as well as sites for necessary processing information, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from leukoimmunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al, J. Immunol. 148:1149 (1992).

Once synthesized (chemically or recombinantly), the fibers of the present invention can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, and the like lysinogen. The plasminogen is substantially pure, eg, at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure or purer, eg, free of contaminants such as cellular debris, macromolecules other than the target product, and the like.

drug formulation

Lyophilized formulations can be formed by mixing plasminogen of the desired purity with optional pharmaceutical carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). or aqueous solutions to prepare therapeutic formulations. Acceptable carriers, excipients, stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexanediamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parahydroxybenzoic acid Esters such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues) ; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspartamine, histidine, arginine or lysine; monosaccharides, Disaccharides and other carbohydrates include glucose, mannose, or dextrin; chelating agents such as EDTA; carbohydrates such as sucrose, mannitol, fucose, or sorbitol; salt-forming counterions such as sodium; complex); and/or nonionic surfactants such as TWENTM, PLURONICSTM or polyethylene glycol (PEG). Preferred lyophilized anti-VEGF antibody formulations are described in WO 97/04801, which is incorporated herein by reference.

The formulations of the present invention may also contain more than one active compound as required for the particular condition to be treated, preferably those which complement each other in activity and which do not have side effects with each other. For example, antihypertensive drugs, antiarrhythmic drugs, diabetes drugs, etc.

The plasminogen of the present invention can be encapsulated in microcapsules prepared by techniques such as coacervation or interfacial polymerization, for example, can be placed in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in hydroxymethyl cellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The plasminogen of the present invention for in vivo administration must be sterile. This can be easily achieved by filtration through sterile filters before or after lyophilization and reconstitution.

The plasminogen of the present invention can be prepared as a sustained-release preparation. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers having a shape and containing glycoproteins, such as membranes or microcapsules. Examples of sustained release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981); Langer, Chem. Tech., 12:98-105 (1982)) or poly(vinyl alcohol), polylactide (US Patent 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamic acid (Sidman, et al., Biopolymers 22:547 (1983)), non-degradable ethylene-vinyl acetate (Langer, et al., supra), or degradable lactic acid-glycolic acid copolymers such as Lupron DepotTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene- Vinyl acetate and lactic acid-glycolic acid can continuously release molecules for more than 100 days, while some hydrogels release proteins for shorter periods of time. Rational strategies to stabilize proteins can be devised based on the relevant mechanisms. For example, if the mechanism of aggregation is found to be Intermolecular SS bonds are formed by thiodisulfide exchange, and stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling humidity, using suitable additives, and developing specific polymer matrix compositions.

Administration and Dosage

Administration may be by various means, such as by nasal inhalation, nebulization, nasal or eye drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (eg via the carotid), intramuscular, rectal drug to achieve the administration of the pharmaceutical composition of the present invention.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, and the like. Preservatives and other additives may also be present, such as, antimicrobials, antioxidants, chelating agents, and inert gases, among others.

Dosing regimens will be determined by healthcare professionals based on various clinical factors. As is well known in the medical arts, the dosage for any patient depends on a variety of factors, including the patient's size, body surface area, age, the particular compound to be administered, gender, number and route of administration, general health, and other concomitantly administered drugs. The dosage range of the pharmaceutical composition comprising plasminogen of the present invention may be about 0.0001 to 2000 mg/kg, or about 0.001 to 500 mg/kg (eg, 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 10 mg/kg, 50 mg/kg, etc.) subject body weight. For example, the dose may be 1 mg/kg body weight or 50 mg/kg body weight or in the range of 1-50 mg/kg, or at least 1 mg/kg. Dosages above or below this exemplary range are also contemplated, especially in view of the factors set forth above. Intermediate doses within the above ranges are also included within the scope of the present invention. Subjects may be administered such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. An exemplary dosage schedule includes 0.01-100 mg/kg on consecutive days. Real-time evaluation of therapeutic efficacy and safety is required during the administration of the drug of the present invention.

product or kit

One embodiment of the present invention relates to an article of manufacture or kit comprising plasminogen or plasmin of the present invention useful in the treatment of cardiovascular disease caused by diabetes and related disorders. The article of manufacture preferably includes a container, label or package insert. Suitable containers are bottles, vials, syringes, etc. The container can be made of various materials such as glass or plastic. The container contains a composition effective to treat the disease or disorder of the present invention and has a sterile access port (eg, the container may be an intravenous solution pack or vial containing a stopper penetrable by a hypodermic needle). ). At least one active agent in the composition is plasminogen/plasmin. The label on or attached to the container indicates that the composition is used for the treatment of cardiovascular disease caused by diabetes and related disorders according to the present invention. The article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may further contain other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes. In addition, the article of manufacture comprises a package insert with instructions for use, including, for example, instructing the user of the composition to administer the plasminogen composition to the patient along with other drugs to treat accompanying conditions.

Example

Human plasminogen used in all of the following examples was derived from human donor plasma based on methods described in: Kenneth C Robbins, Louis Summaria, David Elwyn et al. Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin. Journal of Biological Chemistry, 1965, 240(1):541-550;Summaria L, Spitz F, Arzadon L et al. Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins. J Biol Chem. 1976 Jun 25;251(12):3693-9; HAGAN JJ, ABLONDI FB, DE RENZO EC. Purification and biochemical properties of human plasminogen. J Biol Chem. 1960 Apr; 235:1005-10, and process optimization, from human Purified from donor plasma. The purity of plasminogen monomer is >98%.

Example 1 Plasminogen improves spontaneous activity and avoidance behavior in Huntington model mice

Take 6-week-old male C57 mice, weigh all mice 3 days before modeling and conduct a simple open field experiment, observe for 5 min, exclude spontaneous differences in random animals, and finally determine 23 experimental animals, according to the open field experiment medium and small. The total movement distance of the mice The mice were randomly divided into 2 groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were intraperitoneally injected with 125 μL of PBS solution, and the mice in the model group were intraperitoneally injected with 50 mg/kg body weight of 3-Nitropropionic acid (3-NP) solution, twice a day (12 hours apart) for 5 consecutive injections. Day, the establishment of Huntington model ^[1] . 3-NP solution preparation: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg/ml. The second day after the completion of the modeling was designated as the 0th day of administration. All mice were weighed and subjected to an open field experiment. The mice in the model group were randomly divided into 2 groups according to the total exercise distance in the open field experiment. The vehicle control group (9 mice) and the plasminogen group (8 mice), the mice in the vehicle control group were injected with vehicle (10mM sodium citrate solution, pH 7.4) through the tail vein of 0.1ml/mice every day, and the mice in the plasminogen group were given small mice. Mice were injected with human plasminogen by tail vein of 1mg/0.1ml/mice every day, and the administration was continued for 7 days. Open field experiments were performed on the 7th day of dosing.

3-NP is a natural mycotoxin that irreversibly inhibits mitochondrial succinate dehydrogenase, causing neurotoxicity across the blood-brain barrier, leading to neuronal death, mimicking human HD symptoms ^[1] .

open field experiment

During the experiment, the mice were placed in the center of the bottom surface of the open field (40x40x40cm), and images were taken and timed at the same time. The observation lasted for 5 minutes, and each mouse performed 3 experiments. The Smart System is a complete and easy-to-use video tracking system for evaluating the behavior of laboratory animals. It allows to record movement trajectories, activities, specific behaviors (such as rotation, stretching and feeding) and events, and to perform calculations of various analytical parameters. In this experiment, the Smart3.0 system was used to record and analyze the movement of the mice. The parameters included the total moving distance, the border resting time rate, the average moving speed of the central area and the average moving speed of the border. After each experiment, use 70% alcohol to wipe the box to prevent the preference of smell ^[1] .

The design principle of the open field experiment is based on the avoidance of mice, which means that mice are afraid of open, unknown, and potentially dangerous places, so they have the nature of "sticking to the wall". Avoidance was assessed by mouse activity in the surrounding area (four corners and four sides) of the field. Judging from the activity time in the surrounding area, which reflects avoidance, the time is reduced, indicating that the mice are more "adventurous" tendencies. Significantly more active time in the central zone, indicating lower levels of avoidance and anxiety (depression).

total exercise distance

The total movement distance refers to the length of the movement track within the specified test time. The results showed that the blank control group would exercise a certain distance during the experiment; the total movement distance of the mice in the vehicle control group was significantly longer than that in the blank control group; the total movement distance of the mice in the plasminogen group was significantly shorter than that in the vehicle control group, and the statistical difference was significant ( * indicates P<0.05), and the total exercise distance of the plasminogen group was close to that of the blank control group (Figure 1). This indicates that plasminogen can promote the recovery of spontaneous activity in Huntington model mice.

Boundary zone resting time rate

The boundary area is the surrounding area of the wilderness (four corners and four sides). The rate of resting time in the border zone refers to the ratio of the resting time in the border zone to the total resting time (including the resting time in the border zone and the resting time in the central zone). The results showed that the mice in the blank control group had a certain rate of resting time in the border area; the rate of resting time in the border area of the mice in the vehicle control group was significantly lower than that in the blank control group; the rate of resting time in the border area of the mice in the plasminogen group was significantly lower. Compared with the vehicle control group, the statistical difference was extremely significant (*** indicates P<0.001), and the border resting time rate of the plasminogen group was close to that of the blank control group (Figure 2). This indicates that plasminogen can alleviate the anxiety behavior of Huntington model mice.

Average speed of movement in the central area

The central area refers to the central 20x20cm area of the open field. The average exercise speed in the central area refers to the ratio of the total exercise distance in the central area to the total stay time in the central area (including exercise and rest time). The results showed that the mice in the blank control group had a certain movement speed in the central area; the average movement speed in the central area of the mice in the vehicle control group was significantly lower than that in the blank control group; the average movement speed in the central area of the mice in the plasminogen group was significantly greater than that in the vehicle control group. , the statistical difference was significant (* means P<0.05), and the average movement speed of the central area of the plasminogen group was close to that of the blank control group (Figure 3). This indicates that plasminogen can promote the spontaneous activity of Huntington model mice and relieve anxiety behavior.

The average speed of movement in the boundary area

The average speed of movement in the boundary area refers to the ratio of the total movement distance in the boundary area to the total residence time in the boundary area. The results showed that the blank control group had a certain movement speed in the boundary area; the average movement speed of the mice in the vehicle control group was significantly higher than that in the blank control group; the average movement speed of the mice in the plasminogen group was significantly lower than that in the vehicle control group. The difference was significant (* means P<0.05), and the average movement speed of the border zone in the plasminogen group was close to that in the blank control group (Fig. 4). This indicates that plasminogen can promote spontaneous activity and relieve anxiety behavior in Huntington model mice.

Movement track

The results showed that the mice in the blank control group (Figure 5A) had less movement in the central area, more movement in the border area, and regular movement trajectories; compared with the blank control group, the movement and total distance of the central area of the mice in the vehicle group (Figure 5B) increased significantly , the movement trajectory was chaotic and irregular; the plasminogen group (Fig. 5C) mouse central area movement and total movement distance were significantly less than the vehicle group, and the movement trajectory was similar to the blank control group. This indicated that plasminogen could promote the recovery of spontaneous activity and avoidance behavior in Huntington model mice.

Example 2 Plasminogen slows weight loss in Huntington's model mice

Take 6-week-old male C57 mice, weigh all mice 3 days before modeling and conduct a simple open-field experiment, observe for 5 minutes, exclude spontaneous differences in random animals, and finally determine 23 experimental animals, according to the open-field experiment The total exercise distance of the middle-aged mice was randomly divided into 2 groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were intraperitoneally injected with 125 μL of PBS solution, and the mice in the model group were intraperitoneally injected with 50 mg/kg body weight of 3-Nitropropionic acid (3-NP) solution, twice a day (12 hours apart) for 5 consecutive injections. Day, the establishment of Huntington model ^[1] . 3-NP solution preparation: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg/ml. The second day after the completion of the modeling was designated as the 0th day of administration. All mice were weighed and subjected to an open field experiment. The mice in the model group were randomly divided into 2 groups according to the total exercise distance in the open field experiment. The vehicle control group (9 mice) and the plasminogen group (8 mice), the mice in the vehicle control group were injected with vehicle (10mM citric acid-sodium citrate solution, pH7.4) through the tail vein of 0.1ml/mice every day, and the mice were given plasmin. The mice in the original group were injected with human plasminogen by tail vein of 1 mg/0.1 ml/mice every day for 7 consecutive days. The body weight of the mice was measured on the 7th day of administration, and the percentage of body weight on the 7th day and the 0th day was calculated.

Weight loss is a common non-neurological phenotype in Huntington's that is usually progressive and can lead to malnutrition or cachexia ^[5] .

The results showed that the weight percentage of the blank control group was greater than 1, and the weight increased; the weight percentage of the mice in the vehicle control group was smaller than that of the blank control group; the weight percentage of the mice in the plasminogen group was significantly higher than that in the vehicle control group, and the statistical difference was close to significant (P=0.07) (Figure 6). The results suggest that plasminogen can promote the weight gain of Huntington model mice.

Example 3 Plasminogen reduces the hippocampus of Huntington's model mice GFAP Express

Take 6-week-old male C57 mice, weigh all mice 3 days before modeling and conduct a simple open-field experiment, observe for 5 minutes, exclude spontaneous differences in random animals, and finally determine 23 experimental animals, according to the open-field experiment The total exercise distance of the middle-aged mice was randomly divided into 2 groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were intraperitoneally injected with 125 μL of PBS solution, and the mice in the model group were intraperitoneally injected with 50 mg/kg body weight of 3-Nitropropionic acid (3-NP) solution, twice a day (12 hours apart) for 5 consecutive injections. Days, the establishment of the Huntington model ^[1] . 3-NP solution preparation: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg/ml. The second day after the completion of the modeling was designated as the 0th day of administration. All mice were weighed and subjected to an open field experiment. The mice in the model group were randomly divided into 2 groups according to the total exercise distance in the open field experiment. The vehicle control group (9 mice) and the plasminogen group (8 mice), the mice in the vehicle control group were injected with vehicle (10mM citric acid-sodium citrate, pH 7.4) through the tail vein of 0.1ml/mice every day, and the mice were given plasminogen. The mice in the group were injected with human plasminogen by tail vein at 1 mg/0.1 ml/mice every day for 7 consecutive days. On the 7th day of administration, the mice were sacrificed and the hippocampus was fixed in 10% neutral formaldehyde solution for 24-48 hours. The fixed hippocampal tissue was dehydrated in alcohol gradient and cleared with xylene before being embedded in paraffin. The thickness of tissue sections was 3 μm, and the sections were deparaffinized and rehydrated, and washed once with water. Tissue was circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 min, and washed twice with 0.01M PBS for 5 min each. 5% normal goat serum (Vector laboratories, Inc., USA) was blocked for 30 minutes; when the time was up, the goat serum was discarded, rabbit-derived anti-GFAP antibody (ab7260, Abcam) was added dropwise and incubated at 4°C overnight, 0.01M Wash 2 times with PBS, 5 min each. Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated at room temperature for 1 hour, and washed twice with 0.01M PBS for 5 minutes each. The color was developed according to DAB kit (Vector laboratories, Inc., USA), washed with water for 3 times, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. Gradient alcohol dehydration, clear xylene and neutral gum to seal the sections, and the sections were observed under a 400-fold optical microscope.

Glial fibrillary acidic protein (GFAP) is a hallmark intermediate filament protein in astrocytes, which participates in the formation of the cytoskeleton and maintains its tensile strength. Astrocytes are one of the main glial cells in the central nervous system. Studies have reported that Huntington's disease is accompanied by astroglial hyperplasia and enhanced activity, manifested as up-regulation of GFAP expression and cell hypertrophy ^[6-7] .

The results showed that the hippocampus of the mice in the blank control group (Figure 7A) expressed a certain amount of GFAP; the expression of GFAP in the hippocampus of the mice in the vehicle control group (Figure 7B) was significantly higher than that of the blank control group, and the statistical difference was extremely significant (*** indicates P <0.001) (Figure 7D); the expression of GFAP in the hippocampus of mice in the plasminogen group (Figure 7C) was significantly lower than that in the vehicle control group, and the statistical difference was extremely significant, and compared with the expression of GFAP in the hippocampus of mice in the blank control group No statistical difference. The results indicate that plasminogen can reduce the expression of GFAP in the hippocampus of Huntington model mice, inhibit the proliferation of astroglial cells, and have neuroprotective effects.

Example 4 Plasminogen reduces apoptosis in the hippocampus of Huntington's model mice

Take 6-week-old male C57 mice, weigh all mice 3 days before modeling and conduct a simple open-field experiment, observe for 5 minutes, exclude spontaneous differences in random animals, and finally determine 23 experimental animals, according to the open-field experiment The total exercise distance of the middle-aged mice was randomly divided into 2 groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were intraperitoneally injected with 125 μL of PBS solution, and the mice in the model group were intraperitoneally injected with 50 mg/kg body weight of 3-Nitropropionic acid (3-NP) solution, twice a day (12 hours apart) for 5 consecutive injections. Day, the establishment of Huntington model ^[1] . 3-NP solution preparation: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg/ml. The second day after the completion of the modeling was designated as the 0th day of administration. All mice were weighed and subjected to an open field experiment. The mice in the model group were randomly divided into 2 groups according to the total exercise distance in the open field experiment. The vehicle control group (9 mice) and the plasminogen group (8 mice), the mice in the vehicle control group were injected with vehicle (10mM citric acid-sodium citrate solution, pH7.4) through the tail vein of 0.1ml/mice every day, and the mice were given plasmin. The mice in the original group were injected with human plasminogen by tail vein of 1 mg/0.1 ml/mice every day for 7 consecutive days. On the 7th day of administration, the mice were sacrificed and the hippocampus was fixed in 10% neutral formaldehyde solution for 24-48 hours. The fixed hippocampal tissue was dehydrated with alcohol gradient and cleared with xylene, and then embedded in paraffin. The thickness of tissue sections was 3 μm, and the sections were deparaffinized and rehydrated, and washed once with water. Tissue was circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 min, and washed twice with 0.01M PBS for 5 min each. 5% normal goat serum (Vector laboratories, Inc., USA) was blocked for 30 minutes; when the time was up, the goat serum was discarded, and rabbit anti-caspase-3 antibody (BA2142, Boster Biological Technology) was added dropwise and incubated at 4°C Overnight, wash twice with 0.01M PBS for 5 minutes each. Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated at room temperature for 1 hour, and washed twice with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), washed with water for 3 times, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. Gradient alcohol dehydration, clear xylene and neutral gum to seal the sections, and the sections were observed under a 400-fold optical microscope.

The cysteine protease (Caspase) family plays a very important role in the process of mediating apoptosis, among which caspase-3 is a key executive molecule that functions in many pathways of apoptosis signaling. In the 3-NP Huntington model, neuronal apoptosis was increased and caspase-3 expression was upregulated.

The results showed that the hippocampus of the blank control group (Fig. 8A) expressed a certain amount of caspase-3 (marked by arrows), while the expression of caspase-3 was significantly increased in the hippocampus of the vehicle control group (Fig. 8B). The expression of cysteine protease-3 in the hippocampus of the lysinogen group (Fig. 8C) was significantly lower than that of the vehicle control group, and the statistical difference was close to significant (P=0.058) (Fig. 8D). The results suggest that plasminogen can reduce the expression of caspase-3 and reduce the apoptosis of hippocampal cells in Huntington model mice.

Example 5 Plasminogen promotes recovery of Nissl bodies in cerebellar neurons of Huntington model mice

Take 6-week-old male C57 mice, weigh all mice 3 days before modeling and conduct a simple open-field experiment, observe for 5 minutes, exclude spontaneous differences in random animals, and finally determine 23 experimental animals, according to the open-field experiment The total exercise distance of the middle-aged mice was randomly divided into 2 groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were intraperitoneally injected with 125 μL of PBS solution, and the mice in the model group were intraperitoneally injected with 50 mg/kg body weight of 3-Nitropropionic acid (3-NP) solution, twice a day (12 hours apart) for 5 consecutive injections. Day, the establishment of Huntington model ^[1] . 3-NP solution preparation: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg/ml. The second day after the completion of the modeling was designated as the 0th day of administration. All mice were weighed and subjected to an open field experiment. The mice in the model group were randomly divided into 2 groups according to the total exercise distance in the open field experiment. The vehicle control group (9 mice) and the plasminogen group (8 mice), the mice in the vehicle control group were injected with vehicle (10mM citric acid-sodium citrate solution, pH7.4) through the tail vein of 0.1ml/mice every day, and the mice were given plasmin. The mice in the original group were injected with human plasminogen by tail vein of 1 mg/0.1 ml/mice every day for 7 consecutive days. On the 7th day of administration, the mice were sacrificed and the cerebellum was fixed in 10% neutral formaldehyde solution for 24-48 hours. The fixed cerebellar tissue was dehydrated with alcohol gradient and cleared with xylene, and then embedded in paraffin. Tissue sections were 3 μm thick, dewaxed to water, and stained with 0.5% toluidine blue staining solution. Dehydrated with graded alcohol, transparent with xylene, and mounted with neutral gum. Under an optical microscope, observe and take pictures.

Nissl bodies are one of the characteristic structures of neurons, and are the synthesis sites of structural and functional proteins of neurons. The number and distribution of them are closely related to the functional state of neurons. The main chemical components of Nissl body are ribonucleic acid and protein, which is called extranuclear chromatin and has affinity with basic dyes such as toluidine blue, thionine and tar violet, among which toluidine blue staining is the most commonly used display. The traditional method of Nisslite ^[8] . When neurological disorders occur, the state of Nissl bodies can be abnormal.

The results showed that there were a certain number of Nissl bodies (marked by arrows) in the cerebellar neurons of the blank control group (Fig. 9A); the number of Nissl bodies in the cerebellar neurons of the vehicle control group (Fig. 9B) was significantly higher than that of the blank control group; The number of Nissl bodies in the cerebellar neurons of the original group (Fig. 9C) was not significantly different from that of the blank control group, but was significantly less than that of the vehicle control group, and the difference was statistically significant (* indicates P<0.05) (Fig. 9D). These results indicate that plasminogen can promote the recovery of Nissl bodies in the cerebellar neurons of Huntington model mice.

Example 6 Plasminogen promotes recovery of Nissl bodies in hippocampal neurons of Huntington model mice

Take 6-week-old male C57 mice, weigh all mice 3 days before modeling and conduct a simple open-field experiment, observe for 5 minutes, exclude spontaneous differences in random animals, and finally determine 23 experimental animals, according to the open-field experiment The total exercise distance of the middle-aged mice was randomly divided into 2 groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were intraperitoneally injected with 125 μL of PBS solution, and the mice in the model group were intraperitoneally injected with 50 mg/kg body weight of 3-Nitropropionic acid (3-NP) solution, twice a day (12 hours apart) for 5 consecutive injections. Day, the establishment of Huntington model ^[1] . 3-NP solution preparation: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg/ml. The second day after the completion of the modeling was designated as the 0th day of administration. All mice were weighed and subjected to an open field experiment. The mice in the model group were randomly divided into 2 groups according to the total exercise distance in the open field experiment. The vehicle control group (9 mice) and the plasminogen group (8 mice), the mice in the vehicle control group were injected with vehicle (10mM citric acid-sodium citrate solution, pH7.4) through the tail vein of 0.1ml/mice every day, and the mice were given plasmin. The mice in the original group were injected with human plasminogen by tail vein of 1 mg/0.1 ml/mice every day for 7 consecutive days. On the 7th day of administration, the mice were sacrificed and the hippocampus was fixed in 10% neutral formaldehyde solution for 24-48 hours. The fixed hippocampal tissue was dehydrated with alcohol gradient and cleared with xylene, and then embedded in paraffin. The thickness of tissue sections was 3 μm, and after dewaxing to water, stained with 0.4% tar violet staining solution (manufacturer: Sinopharm Chemical Reagent Co., Ltd., batch number: 20181019) (pH=3). Dehydrated with graded alcohol, transparent with xylene, and mounted with neutral gum. Sections were observed and photographed under a 400x optical microscope.

Tar violet dyeing uses tar violet as the core dye. Tar violet has a photosensitive effect and can well display the changes of Nissl body.

The results showed that there were a certain amount of Nissl bodies (marked by arrows) in the left hippocampal neurons of the mice in the blank control group (Fig. 10A); the number of Nissl bodies in the left hippocampal neurons of the vehicle group (Fig. 10B) was significantly more than that of the blank control group (Fig. 10B). In the control group, the number of Nissl bodies in the left hippocampus of mice in the plasminogen group (Fig. 10C) was significantly less than that in the vehicle group, and the difference was statistically significant (* indicates P<0.05) (Fig. 10D). The results suggest that plasminogen can promote the recovery of the number of Nissl bodies in hippocampal neurons.

Example 7 Plasminogen promotes recovery of Nissl bodies in striatal neurons in Huntington's model mice

Take 6-week-old male C57 mice, weigh all mice 3 days before modeling and conduct a simple open-field experiment, observe for 5 minutes, exclude spontaneous differences in random animals, and finally determine 23 experimental animals, according to the open-field experiment The total exercise distance of the middle-aged mice was randomly divided into 2 groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were intraperitoneally injected with 125 μL of PBS solution, and the mice in the model group were intraperitoneally injected with 50 mg/kg body weight of 3-Nitropropionic acid (3-NP) solution, twice a day (12 hours apart) for 5 consecutive injections. Day, the establishment of Huntington model ^[1] . 3-NP solution preparation: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg/ml. The second day after the completion of the modeling was designated as the 0th day of administration. All mice were weighed and subjected to an open field experiment. The mice in the model group were randomly divided into 2 groups according to the total exercise distance in the open field experiment. The vehicle control group (9 mice) and the plasminogen group (8 mice), the mice in the vehicle control group were injected with vehicle (10mM citric acid-sodium citrate solution, pH7.4) through the tail vein of 0.1ml/mice every day, and the mice were given plasmin. The mice in the original group were injected with human plasminogen by tail vein of 1 mg/0.1 ml/mice every day for 7 consecutive days. On the 7th day of administration, the mice were sacrificed and the striatum was fixed in 10% neutral formaldehyde solution for 24-48 hours. The fixed striatal tissue was dehydrated with alcohol gradient and cleared with xylene before paraffin-embedding. The tissue sections were 3 μm thick, dewaxed to water, and stained with 0.4% tar violet staining solution (pH=3). Dehydrated with graded alcohol, transparent with xylene, and mounted with neutral gum. Sections were observed and photographed under a 400x optical microscope.

The results showed that there were a certain amount of Nissl bodies (marked by arrows) in the striatal neurons of the mice in the blank control group (Fig. 11A); the number of Nissl bodies in the striatal neurons of the mice in the vehicle group (Fig. 11B) was significantly less than that of the blank control group (Fig. 11B). In the control group, the number of Nissl bodies in the striatal neurons of mice in the plasminogen group (Fig. 11C) was significantly higher than that in the vehicle group, and the difference was statistically significant (* indicates P<0.05) (Fig. 11D). These results suggest that plasminogen can promote the recovery of the number of striatal neurons in Nissl bodies.

Example 8 Plasminogen reduces hippocampal injury in Huntington's model mice

Take 6-week-old male C57 mice, weigh all mice 3 days before modeling and conduct a simple open-field experiment, observe for 5 minutes, exclude spontaneous differences in random animals, and finally determine 23 experimental animals, according to the open-field experiment The total exercise distance of the middle-aged mice was randomly divided into 2 groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were intraperitoneally injected with 125 μL of PBS solution, and the mice in the model group were intraperitoneally injected with 50 mg/kg body weight of 3-Nitropropionic acid (3-NP) solution, twice a day (12 hours apart) for 5 consecutive injections. Day, the establishment of Huntington model ^[1] . 3-NP solution preparation: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg/ml. The second day after the completion of the modeling was designated as the 0th day of administration. All mice were weighed and subjected to an open field experiment. The mice in the model group were randomly divided into 2 groups according to the total exercise distance in the open field experiment. The vehicle control group (9 mice) and the plasminogen group (8 mice), the mice in the vehicle control group were injected with vehicle (10mM citric acid-sodium citrate solution, pH7.4) through the tail vein of 0.1ml/mice every day, and the mice were given plasmin. The mice in the original group were injected with human plasminogen by tail vein of 1 mg/0.1 ml/mice every day for 7 consecutive days. On the 7th day of administration, the mice were sacrificed and the hippocampus was fixed in 10% neutral formaldehyde solution for 24-48 hours. The fixed hippocampal tissue was dehydrated in alcohol gradient and cleared with xylene before being embedded in paraffin. The thickness of the slices was 4 μm. The slices were dewaxed, rehydrated and stained with hematoxylin and eosin (HE staining). After differentiation with 1% hydrochloric acid alcohol, ammonia water returned to blue, and the slices were dehydrated and dehydrated in an alcohol gradient. The slices were observed under a 200-fold optical microscope.

The results showed that the overall shape of the hippocampus of the mice in the blank control group (Fig. 12A-C) was normal and the structure was complete; in the vehicle control group (Fig. 12D-F), the neurons in the CA1 region of the hippocampus had disordered arrangement and neuronal atrophy (thin arrows). mark), cell degeneration and necrosis, loose and edema of the surrounding brain tissue (triangle mark), multiple neuronal nuclei in CA3 area with pyknosis and hyperchromatic staining (thick arrow mark); The morphology of the area was recovered to some extent, and the nuclei of individual neurons in the CA3 and DG areas were partially condensed and heavily stained. These results indicate that plasminogen can repair hippocampal injury in Huntington model mice.

Example 9 Plasminogen reduces striatal injury in Huntington's model mice

Take 6-week-old male C57 mice, weigh all mice 3 days before modeling and conduct a simple open-field experiment, observe for 5 minutes, exclude spontaneous differences in random animals, and finally determine 23 experimental animals, according to the open-field experiment The total exercise distance of the middle-aged mice was randomly divided into 2 groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were intraperitoneally injected with 125 μL of PBS solution, and the mice in the model group were intraperitoneally injected with 50 mg/kg body weight of 3-Nitropropionic acid (3-NP) solution, twice a day (12 hours apart) for 5 consecutive injections. Day, the establishment of Huntington model ^[1] . 3-NP solution preparation: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg/ml. The second day after the completion of the modeling was designated as the 0th day of administration. All mice were weighed and subjected to an open field experiment. The mice in the model group were randomly divided into 2 groups according to the total exercise distance in the open field experiment. The vehicle control group (9 mice) and the plasminogen group (8 mice), the mice in the vehicle control group were injected with vehicle (10mM citric acid-sodium citrate solution, pH7.4) through the tail vein of 0.1ml/mice every day, and the mice were given plasmin. The mice in the original group were injected with human plasminogen by tail vein of 1 mg/0.1 ml/mice every day for 7 consecutive days. On the 7th day of administration, the mice were sacrificed and the striatum was fixed in 10% neutral formaldehyde solution for 24-48 hours. The fixed striatal tissue was dehydrated with alcohol gradient and cleared with xylene before paraffin-embedding. The thickness of the slices was 3 μm. The slices were dewaxed, rehydrated and stained with hematoxylin and eosin (HE staining). After differentiation with 1% hydrochloric acid alcohol, ammonia water returned to blue, and the slices were dehydrated and dehydrated in an alcohol gradient. The slices were observed under a 200-fold optical microscope.

The results showed that the striatum of mice in the blank control group (Fig. 13A) was normal in structure; the striatal neurons of the mice in the vehicle group (Fig. 13B) were degenerated, some nuclei were vacuolated (marked by arrows), and the arrangement of brain cells was disordered; The overall structure of the striatum of mice in the plasminogen group (Fig. 13C) was significantly improved, which was similar to the blank control group. The results indicate that plasminogen can repair striatal injury in Huntington model mice.

Example 10 Plasminogen ameliorates olfactory nodule injury in Huntington model mice

Take 6-week-old male C57 mice, weigh all mice 3 days before modeling and conduct a simple open-field experiment, observe for 5 minutes, exclude spontaneous differences in random animals, and finally determine 23 experimental animals, according to the open-field experiment The total exercise distance of the middle-aged mice was randomly divided into 2 groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were intraperitoneally injected with 125 μL of PBS solution, and the mice in the model group were intraperitoneally injected with 50 mg/kg body weight of 3-Nitropropionic acid (3-NP) solution, twice a day (12 hours apart) for 5 consecutive injections. Days, the establishment of the Huntington model ^[1] . 3-NP solution preparation: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg/ml. The second day after the completion of the modeling was designated as the 0th day of administration. All mice were weighed and subjected to an open field experiment. The mice in the model group were randomly divided into 2 groups according to the total exercise distance in the open field experiment. The vehicle control group (9 mice) and the plasminogen group (8 mice), the mice in the vehicle control group were injected with vehicle (10mM citric acid-sodium citrate solution, pH7.4) through the tail vein of 0.1ml/mice every day, and the mice were given plasmin. The mice in the original group were injected with human plasminogen by tail vein of 1 mg/0.1 ml/mice every day for 7 consecutive days. On the seventh day of administration, the mouse brain tissue was sacrificed and fixed in 10% neutral formaldehyde solution for 24-48 hours. The fixed brain tissue was dehydrated with alcohol gradient and cleared with xylene before being embedded in paraffin. The thickness of the slices was 3 μm. The slices were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining). After differentiation with 1% hydrochloric acid alcohol, the ammonia water returned to blue and the slices were dehydrated and dehydrated in an alcohol gradient. The slices were positioned and observed under a 200-fold optical microscope. condition.

It has been reported that the olfactory nerve of Huntington model mice is damaged, and Huntington patients are accompanied by olfactory deficits ^[9] .

The results showed that the olfactory nodules in the brain of the mice in the blank control group (Fig. 14A) had a normal structure and morphology, while the olfactory nodules of the vehicle group (Fig. 14B) had severe glial cell infiltration (marked by arrows), and the plasminogen group (Fig. 14C) The mouse olfactory tubercle glial cell infiltration was significantly less than the vehicle group. These results suggest that plasminogen can improve brain olfactory nodule injury in Huntington model mice.

Example 11 Plasminogen promotes the hippocampus of Huntington's model mice BDNF expression

Take 6-week-old male C57 mice, weigh all mice 3 days before modeling and conduct a simple open-field experiment, observe for 5 minutes, exclude spontaneous differences in random animals, and finally determine 23 experimental animals, according to the open-field experiment The total exercise distance of the middle-aged mice was randomly divided into 2 groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were intraperitoneally injected with 125 μL of PBS solution, and the mice in the model group were intraperitoneally injected with 50 mg/kg body weight of 3-Nitropropionic acid (3-NP) solution, twice a day (12 hours apart) for 5 consecutive injections. Day, the establishment of Huntington model ^[1] . 3-NP solution preparation: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg/ml. The second day after the completion of the modeling was designated as the 0th day of administration. All mice were weighed and subjected to an open field experiment. The mice in the model group were randomly divided into 2 groups according to the total exercise distance in the open field experiment. The vehicle control group (9 mice) and the plasminogen group (8 mice), the mice in the vehicle control group were injected with vehicle (10mM citric acid-sodium citrate solution, pH7.4) through the tail vein of 0.1ml/mice every day, and the mice were given plasmin. The mice in the original group were injected with human plasminogen by tail vein of 1 mg/0.1 ml/mice every day for 7 consecutive days. On the 7th day of administration, the mice were sacrificed and the hippocampus was fixed in 10% neutral formaldehyde solution for 24-48 hours. The fixed hippocampal tissue was dehydrated with alcohol gradient and cleared with xylene, and then embedded in paraffin. The thickness of tissue sections was 3 μm, and the sections were deparaffinized and rehydrated, and washed once with water. Tissue was circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 min, and washed twice with 0.01M PBS for 5 min each. 5% normal goat serum (Vector laboratories, Inc., USA) was blocked for 30 minutes; when the time was up, the goat serum was discarded, and rabbit-derived anti-BNDF antibody (PB9075, Boster Biological Technology) was added dropwise and incubated at 4°C overnight. Wash twice with 0.01M PBS for 5 minutes each. Goat anti-rabbit IgG (HRP) antibody (Vector laboratories, MP-7451) was incubated with secondary antibody at room temperature for 30 minutes and washed twice with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), washed with water for 3 times, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. Gradient alcohol dehydration, clear xylene and neutral gum to seal the sections, and the sections were observed under a 400-fold optical microscope.

BDNF (brain-derived neurotrophic factor) is a protein synthesized in the brain, which is widely distributed in the central nervous system. development plays an important role. BDNF can prevent neuronal injury and death, improve the pathological state of neurons, and promote the regeneration and differentiation of injured neurons and other biological effects ^[10] .

The results showed that the right hippocampus of the mice in the blank control group (Figure 15A) expressed a certain amount of BDNF (marked by arrows), and the expression of BDNF in the right hippocampus of the mice in the vehicle group (Figure 15B) was slightly higher than that of the blank control group. The expression of BDNF in the right hippocampus of mice in the original group (Fig. 15C) was significantly higher than that in the vehicle group, and the statistical difference was close to significant (P=0.07) (Fig. 15D). The results suggest that injury can stimulate the expression of BDNF, and plasminogen can further promote the expression of BDNF, thereby promoting the repair of hippocampal injury in Huntington model mice.

Example 12 Plasminogen promotes striatum in Huntington's model mice BDNF expression

Take 6-week-old male C57 mice, weigh all mice 3 days before modeling and conduct a simple open-field experiment, observe for 5 minutes, exclude spontaneous differences in random animals, and finally determine 23 experimental animals, according to the open-field experiment The total exercise distance of the middle-aged mice was randomly divided into 2 groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were intraperitoneally injected with 125 μL of PBS solution, and the mice in the model group were intraperitoneally injected with 50 mg/kg body weight of 3-Nitropropionic acid (3-NP) solution, twice a day (12 hours apart) for 5 consecutive injections. Day, the establishment of Huntington model ^[1] . 3-NP solution preparation: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg/ml. The second day after the completion of the modeling was designated as the 0th day of administration. All mice were weighed and subjected to an open field experiment. The mice in the model group were randomly divided into 2 groups according to the total exercise distance in the open field experiment. The vehicle control group (9 mice) and the plasminogen group (8 mice), the mice in the vehicle control group were injected with vehicle (10mM citric acid-sodium citrate solution, pH7.4) through the tail vein of 0.1ml/mice every day, and the mice were given plasmin. The mice in the original group were injected with human plasminogen by tail vein of 1 mg/0.1 ml/mice every day for 7 consecutive days. On the 7th day of administration, the mice were sacrificed and the striatum was fixed in 10% neutral formaldehyde solution for 24-48 hours. The fixed striatal tissue was dehydrated with alcohol gradient and cleared with xylene before paraffin-embedding. The thickness of tissue sections was 3 μm, and the sections were deparaffinized and rehydrated, and washed once with water. Tissue was circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 min, and washed twice with 0.01M PBS for 5 min each. 5% normal goat serum (Vector laboratories, Inc., USA) was blocked for 30 minutes; when the time was up, the goat serum was discarded, and rabbit-derived anti-BNDF antibody (PB9075, Boster Biological Technology) was added dropwise and incubated at 4°C overnight. Wash twice with 0.01M PBS for 5 minutes each. Goat anti-rabbit IgG (HRP) antibody (Vector laboratories, MP-7451) was incubated with secondary antibody at room temperature for 30 minutes and washed twice with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), washed with water for 3 times, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. Gradient alcohol dehydration, clear xylene and neutral gum to seal the sections, and the sections were observed under a 400-fold optical microscope.

The results showed that the striatum of mice in the blank control group (Figure 16A) expressed a certain amount of BDNF (marked by arrows), and the expression of BDNF in the striatum of mice in the vehicle group (Figure 16B) was slightly higher than that of the blank control group. The expression of striatal BDNF in the original group (Fig. 16C) was significantly higher than that in the vehicle group, and the statistical difference was close to significant (P=0.1) (Fig. 16D). The results suggest that injury can stimulate the expression of BDNF, and plasminogen can further promote the expression of BDNF, thereby promoting the repair of striatal injury in Huntington model mice.

Example 13 Plasminogen promotes striatal remyelination in Huntington's model mice

Take 6-week-old male C57 mice, weigh all mice 3 days before modeling and conduct a simple open-field experiment, observe for 5 minutes, exclude spontaneous differences in random animals, and finally determine 23 experimental animals, according to the open-field experiment The total exercise distance of the middle-aged mice was randomly divided into 2 groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were intraperitoneally injected with 125 μL of PBS solution, and the mice in the model group were intraperitoneally injected with 50 mg/kg body weight of 3-Nitropropionic acid (3-NP) solution, twice a day (12 hours apart) for 5 consecutive injections. Day, the establishment of Huntington model ^[1] . 3-NP solution preparation: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg/ml. The second day after the completion of the modeling was designated as the 0th day of administration. All mice were weighed and subjected to an open field experiment. The mice in the model group were randomly divided into 2 groups according to the total exercise distance in the open field experiment. The vehicle control group (9 mice) and the plasminogen group (8 mice), the mice in the vehicle control group were injected with vehicle (10mM citric acid-sodium citrate solution, pH7.4) through the tail vein of 0.1ml/mice every day, and the mice were given plasmin. The mice in the original group were injected with human plasminogen by tail vein of 1 mg/0.1 ml/mice every day for 7 consecutive days. On the 7th day of administration, the mice were sacrificed and the striatum was fixed in 10% neutral formaldehyde solution for 24-48 hours. The fixed striatal tissue was dehydrated with alcohol gradient and cleared with xylene before paraffin-embedding. The thickness of the spinal cord tissue cross-section was 4 μm. After dewaxing to water, LFB staining was performed with myelin staining solution. Dehydrated with graded alcohol, transparent with xylene, and mounted with neutral gum. Under an optical microscope, observe and take pictures.

LFB (luxol fast blue) staining uses fast blue staining to stain myelin, which is an effective method to study the localization of the corticospinal tract, myelin lesions, injury and morphological observation of regeneration and repair ^[11-12] .

The results showed that the striatal myelin structure of the mice in the blank control group (Fig. 17) was normal; the amount of myelin sheath proteins in the striatum of the mice in the vehicle group (Fig. 17B) decreased, and cavitary degeneration (marked by arrows) was seen; The amount of myelin protein in the striatum of mice in the zymogen group (Fig. 17C) was significantly higher than that in the vehicle group, and the statistical difference was close to significant (P=0.09) (Fig. 17D). These results suggest that plasminogen can promote the formation of striatal myelin protein in Huntington model mice.

references

[1] Wang L , Wang J , Yang L , et al. Effect of Praeruptorin C on 3-nitropropionic acid induced Huntington’s disease-like symptoms in mice[J]. Biomedicine & Pharmacotherapy, 2017, 86:81-87.

[2] Marchei P , Diverio S , Falocci N , et al. Breed differences in behavioural development in kittens[J]. Physiology & Behavior, 2009, 96(4-5):522-531.

[3] Jacob W, Yassouridis A, Marsicano G, et al. Endocannabinoids render exploratory behaviour largely independent of the test aversiveness: role of glutamatergic transmission. [J]. Genes Brain & Behavior, 2010, 8(7):685-698 .

[4] Walz K, Spencer C, Kaasik K, et al. Behavioral characterization of mouse models for Smith-Magenis syndrome and dup(17)(p11.2p11.2)[J]. Human Molecular Genetics, 2004, 13(4 ):367.

[5] Aziz N A , Burg J M M V D , Landwehrmeyer G B , et al. Weight loss in Huntington disease increases with higher CAG repeat number[J]. Neurology, 2009, 73(7):1506.

[6] Hol EM, Pekny M. Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system.[J]. Current Opinion in Cell Biology, 2015, 32(1):121-130 .

[7] Vagner T, Dvorzhak A, Wójtowicz AM, et al. Systemic application of AAV vectors targeting GFAP-expressing astrocytes in Z-Q175-KI Huntington's disease mice.[J]. Molecular & Cellular Neuroscience, 2016, 77:76- 86.

[8] Xie J X, Feng Y, Yuan J M, et al. Positive effects of bFGF modified rat amniotic epithelial transplantation cells on transected rat optic nerve[J]. Plos One, 2014, 10(3):e0119119.

[9] Kohl Z, Regensburger M, Aigner R, et al. Impaired adult olfactory bulb neurogenesis in the R6/2 mouse model of Huntington's disease[J]. BMC Neuroscience,11,1(2010-09-13), 2010, 11(1):114.

[10] Hyman C, Hofer M, Barde Y A, et al. BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra[J]. Nature, 1991, 350(6315):230-232.

[11] Sun SW, Liang HF, Trinkaus K et al. Noninvasive detection of cuprizone induced axonal damage and demyelination in the mouse corpus callosum. Magn Reson Med. 2006 Feb;55(2):302-8.

[12] Vallières N1, Berard JL, David S et al. Systemic injections of lipopolysaccharide accelerates myelin phagocytosis during Wallerian degeneration in the injured mouse spinal cord. Glia. 2006 Jan 1;53(1):103-13.

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Figure 1 Statistical results of the total movement distance in the open field test of Huntington model mice after administration of plasminogen for 7 days. The results showed that the mice in the blank control group exercised for a certain distance during the experiment; the total exercise distance of the mice in the vehicle control group was significantly longer than that in the blank control group; the total exercise distance of the mice in the plasminogen group was significantly shorter than that in the vehicle control group, with statistical differences Significant (* means P<0.05), and the total exercise distance of the plasminogen group was close to that of the blank control group. It is suggested that plasminogen can promote the recovery of spontaneous activity in Huntington model mice.

Figure 2 Statistical results of the resting time rate at the boundary of the open field experiment in Huntington model mice 7 days after administration of plasminogen. The results showed that the mice in the blank control group had a certain rate of resting time in the border area; the rate of resting time in the border area of the mice in the vehicle control group was significantly lower than that in the blank control group; the rate of resting time in the border area of the mice in the plasminogen group was significantly lower. It was greater than that of the vehicle control group, and the statistical difference was extremely significant (*** indicates P<0.001), and the border resting time rate of the plasminogen group was close to that of the blank control group. It is suggested that plasminogen can alleviate the anxiety behavior of Huntington model mice.

Figure 3 Statistical results of the average movement speed of the central area of the Huntington model mice in the open field experiment after administration of plasminogen for 7 days. The results showed that the mice in the blank control group had a certain movement speed in the central area; the average movement speed in the central area of the mice in the vehicle control group was significantly lower than that in the blank control group; the average movement speed in the central area of the mice in the plasminogen group was significantly greater than that in the vehicle control group. , the statistical difference was significant (* means P<0.05), and the movement speed of the central area of the plasminogen group was close to that of the blank control group. It is suggested that plasminogen can promote the recovery of spontaneous activity and avoidance behavior in Huntington model mice, and relieve anxiety behavior.

Fig. 4 Statistical results of the average movement speed in the boundary zone of the Huntington model mice in the open field experiment after administration of plasminogen for 7 days. The results showed that the blank control group had a certain movement speed in the boundary area; the average movement speed of the mice in the vehicle control group was significantly higher than that in the blank control group; the average movement speed of the mice in the plasminogen group was significantly lower than that in the vehicle control group. The difference was significant (* means P<0.05), and the average movement speed of the border zone in the plasminogen group was close to that in the blank control group. It is suggested that plasminogen can promote the recovery of spontaneous activity and avoidance behavior in Huntington model mice, and relieve anxiety behavior.

Figures 5A-C are diagrams of locomotion trajectories in the open field experiment of Huntington model mice after administration of plasminogen for 7 days. A is the blank control group, B is the vehicle control group, and C is the plasminogen administration group. The results showed that the mice in the blank control group had less movement in the central area, more movement in the boundary area, and regular movement trajectories; compared with the blank control group, the movement of the mice in the vehicle group increased significantly in the central area and total movement distance, and the movement trajectories were chaotic and irregular; The movement and total movement distance in the central area of the mice in the lysinogen group were significantly less than those in the vehicle group, and the movement trajectory was similar to that in the blank control group. It is suggested that plasminogen can promote the recovery of spontaneous activity and avoidance behavior in Huntington model mice.

Figure 6 Calculation results of the percentage of body weight of Huntington model mice on day 7 and day 0 after administration of plasminogen for 7 days. The results showed that the weight percentage of the blank control group was greater than 1, and the weight increased; the weight percentage of the mice in the vehicle control group was smaller than that of the blank control group; the weight percentage of the mice in the plasminogen group was significantly higher than that in the vehicle control group, and the statistical difference was close to significant (P=0.07). The results suggest that plasminogen can promote the weight gain of Huntington model mice.

7A-D GFAP immunohistochemical staining results of the hippocampus of Huntington model mice after administration of plasminogen for 7 days. A is the blank control group, B is the vehicle control group, C is the plasminogen-administered group, and D is the average optical density quantitative analysis result. The results showed that the hippocampus of the mice in the blank control group expressed a certain amount of GFAP; the expression of GFAP in the hippocampus of the mice in the vehicle control group was significantly higher than that in the blank control group, and the statistical difference was extremely significant (*** indicates P<0.001); The expression of GFAP in the hippocampus of the original group of mice was significantly lower than that of the vehicle control group, and the statistical difference was extremely significant, and there was no statistical difference in the expression of GFAP in the hippocampus of the mice in the blank control group. The results suggest that plasminogen can reduce the expression of GFAP in the hippocampus of Huntington model mice, inhibit the proliferation of astroglial cells, and promote the recovery of hippocampal injury.

Figure 8 A-D immunohistochemical staining results of caspase-3 in the hippocampus of Huntington model mice after administration of plasminogen for 7 days. A is the blank control group, B is the vehicle control group, C is the plasminogen-administered group, and D is the average optical density quantitative analysis result. The results showed that a certain amount of cysteine protease-3 (capase-3) (marked by arrow) was expressed in the hippocampus of the blank control group, and the expression of cysteine protease-3 was significantly increased in the hippocampus of the vehicle control group. The expression of cysteine protease-3 in the hippocampus of the group was significantly lower than that of the vehicle control group, and the statistical difference was close to significant (P=0.058). The results suggest that plasminogen can reduce the expression of caspase-3 and reduce the apoptosis of hippocampal cells in Huntington model mice.

Figure 9A-D results of toluidine blue staining of the cerebellum of Huntington model mice after administration of plasminogen for 7 days. A is the blank control group, B is the vehicle control group, C is the plasminogen-administered group, and D is the result of the quantitative analysis of cerebellar Nissl bodies. The results showed that there were a certain number of Nissl bodies in the cerebellar neurons of the blank control group (marked by arrows); the number of Nissl bodies in the cerebellar neurons of the vehicle control group was significantly higher than that of the blank control group; the cerebellar neurons of mice in the plasminogen group were treated with Nissl bodies. There was no significant difference in the amount of tensites with the blank control group, but it was significantly less than that in the solvent control group, and the difference was statistically significant (* means P<0.05). The results suggest that plasminogen can promote the recovery of the number of Nissl bodies in the cerebellar neurons of Huntington model mice.

Figures 10A-D 7 days after administration of plasminogen, the results of tar violet staining of the left hippocampus of Huntington model mice. A is the blank control group, B is the vehicle control group, C is the plasminogen-administered group, and D is the analysis result of the number of Nissl bodies in the hippocampus. The results showed that a certain amount of Nissl bodies (marked by arrows) existed in the left hippocampal neurons of the mice in the blank control group; the number of Nissl bodies in the left hippocampal neurons of the mice in the vehicle group was significantly higher than that in the blank control group; The number of Nissl bodies in the left hippocampus of mice in the group was significantly less than that in the vehicle group, and the difference was statistically significant (* means P<0.05). The results suggest that plasminogen can promote the recovery of the number of Nissl bodies in hippocampal neurons.

Figure 11A-D results of tar violet staining of striatum of Huntington model mice after administration of plasminogen for 7 days. A is the blank control group, B is the vehicle control group, C is the plasminogen-administered group, and D is the number of striatal Nissl bodies. The results showed that there were a certain amount of Nissl bodies (marked by arrows) in the striatal neurons of the mice in the blank control group; the number of Nissl bodies in the striatal neurons of the mice in the vehicle group was significantly less than that in the blank control group; The number of Nissl bodies in striatal neurons of mice in the group was significantly more than that in the vehicle group, and the difference was statistically significant (* means P<0.05). These results suggest that plasminogen can promote the recovery of the number of striatal neurons in Nissl bodies.

12A-I H&E staining results of the hippocampus of Huntington model mice 7 days after administration of plasminogen. A, B and C are blank control groups, D, E and F are vehicle control groups, and G, H, I are plasminogen-administered groups. The results showed that the overall shape of the hippocampus of the mice in the blank control group was normal and the structure was complete; the CA1 region of the hippocampus of the mice in the vehicle control group had disordered arrangement of neurons, atrophy of nerve cells (marked by thin arrows), cell degeneration and necrosis, and loose and edema of the surrounding brain tissue. (marked by a triangle), multiple neuronal nuclei in the CA3 area were pyknoticed and heavily stained (marked by thick arrows); the morphology of each area of the hippocampus in the plasminogen group was restored, and there were individual neuronal nuclei in the CA3 and DG areas. , heavily dyed. These results suggest that plasminogen can repair hippocampal injury in Huntington model mice.

Figure 13A-C Results of H&E staining of striatum in Huntington model mice 7 days after plasminogen administration. A is the blank control group, B is the vehicle control group, and C is the plasminogen administration group. The results showed that the striatal morphological structure of the mice in the blank control group was normal; the striatal neurons of the mice in the vehicle group were degenerated, some nuclei were vacuolated (marked by arrows), and the arrangement of brain cells was disordered; the mice in the plasminogen group were The overall structure of the striatum was significantly improved, similar to the blank control group. The results suggest that plasminogen can repair striatal injury in Huntington model mice.

14A-C H&E staining results of olfactory nodules in Huntington model mice 7 days after administration of plasminogen. A is the blank control group, B is the vehicle control group, and C is the plasminogen administration group. The results showed that the morphology and structure of olfactory nodules in the brains of the mice in the blank control group were normal, the olfactory nodules in the vehicle group had severe glial cell infiltration (marked by arrows), and the infiltration of glial cells in the olfactory nodules of the mice in the plasminogen group was significantly less. in the solvent group. These results suggest that plasminogen can improve brain olfactory nodule injury in Huntington model mice.

Figure 15A-D Results of immunohistochemical staining of BDNF in the hippocampus of Huntington model mice after administration of plasminogen for 7 days. A is the blank control group, B is the vehicle control group, C is the plasminogen administration group, and D is the quantitative analysis result of the average optical density. The results showed that the right hippocampus of the mice in the blank control group expressed a certain amount of BDNF (marked by arrows), and the expression of BDNF in the right hippocampus of the mice in the vehicle group was slightly higher than that in the blank control group. The expression was significantly higher than that in the vehicle group, and the statistical difference was close to significant (P=0.07). The results suggest that injury can stimulate the expression of BDNF, and plasminogen can further promote the expression of BDNF, thereby promoting the repair of hippocampal injury in Huntington model mice.

Figure 16A-D Results of immunohistochemical staining of striatal BDNF in Huntington model mice 7 days after administration of plasminogen. A is the blank control group, B is the vehicle control group, C is the plasminogen-administered group, and D is the average optical density quantitative analysis result. The results showed that the striatum of the mice in the blank control group expressed a certain amount of BDNF (marked by arrows), and the expression of BDNF in the striatum of the mice in the vehicle group was slightly higher than that in the blank control group. The expression was significantly higher than that in the vehicle group, and the statistical difference was close to significant (P=0.1). The results suggest that injury can stimulate the expression of BDNF, and plasminogen can further promote the expression of BDNF, thereby promoting the repair of striatal injury in Huntington model mice.

Figure 17A-D Results of LFB staining in the striatum of Huntington model mice 7 days after administration of plasminogen. A is the blank control group, B is the vehicle control group, C is the plasminogen-administered group, and D is the average optical density quantitative analysis result. The results showed that the striatal myelin structure of the mice in the blank control group was normal; the amount of myelin sheath proteins in the striatum of the mice in the vehicle group decreased, and cavitary degeneration (marked by arrows) was seen in the striatum of the mice in the vehicle group; the striatum of the mice in the plasminogen group The amount of myelin protein was significantly higher than that in the vehicle group, and the statistical difference was close to significant (P=0.09). These results suggest that plasminogen can promote the formation of striatal myelin protein in Huntington model mice.

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Claims (13)

A use of one or more compounds selected from the group consisting of one or more compounds selected from the group consisting of: a component of a plasminogen activation pathway, capable of directly activating plasminogen or by activating fiber Compounds that indirectly activate plasminogen by activating upstream components of the protein lysinogen pathway, compounds that mimic the activity of plasminogen or plasmin, and are capable of upregulating plasminogen or plasminogen activation Agent-expressed compounds, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors. The use according to claim 1, wherein the component of the plasminogen activation pathway is selected from the group consisting of plasminogen, recombinant human plasmin, Lys-plasminogen, Glu-plasminogen Zymogen, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, fibrils Mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta - delta-plasmin, plasminogen activator, tPA and uPA. According to the use of claim 1, the antagonist of the fibrinolysis inhibitor is an inhibitor of PAI-1, complement C1 inhibitor, α2 antiplasmin or α2 macroglobulin, eg an antibody. The use of any one of claims 1-3, wherein the compound has one or more effects on the Huntington's disease subject selected from the group consisting of improving or alleviating movement disorders, improving cognitive dysfunction, slowing weight loss, promoting Repair of damaged neurons, improve neuropsychiatric symptoms, relieve anxiety, reduce hippocampal GFAP expression, reduce hippocampal apoptosis, promote the recovery of cerebellar, hippocampal or striatal neuron Nissl bodies, repair hippocampus, striatum damage to the striatum or olfactory tubercle, promote hippocampal or striatal BDNF expression, and promote striatal remyelination. The use of any one of claims 1-4, wherein the compound is used in combination with one or more other drugs or methods of treatment. The use according to claim 5, wherein the other drug or treatment method is selected from one or more of the following: antidopaminergic drugs, dopamine receptor inhibitors, antipsychotic drugs (such as butylated benzols and phenothiazines ), gamma-aminobutyric acid transferase inhibitors, cell transplantation therapy and gene therapy. The use according to any one of claims 1-6, wherein the compound is plasminogen. The use of any one of claims 1-7, wherein the plasminogen is human full-length plasminogen or a conservatively substituted variant thereof. The use of any one of claims 1-7, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with sequence 2 sequence identity and still possess the lysine-binding or proteolytic activity of plasminogen. The use of any one of claims 1-7, the plasminogen comprising amino acids having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity with sequence 14 sequence and still possess the proteolytic activity of plasminogen. The use according to any one of claims 1 to 7, wherein the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, microplasminogen, microplasminogen, delta-plasminogen The zymogens or their variants that retain the proteolytic activity of plasminogen. The use according to any one of claims 1 to 7, wherein the plasminogen comprises the amino acid sequence shown in the sequence 2, 6, 8, 10, 12 or the amino acid sequence shown in the sequence 2, 6, 8, 10, 12 conservative substitution variants. The use of any one of claims 1-12, wherein the compound is by nasal inhalation, aerosol inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, Intraarterial (eg via the carotid artery) or intramuscular administration.

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